Impact of malathion toxicity on the oxidative stress parameters of the black soldier fly Hermetia illucens (Linnaeus, 1758) (Diptera: Stratiomyidae)

The black soldier fly larvae (BSFL) may serve as a promising tool in the animals feed production industry. The input organic wastes may be contaminated by insecticides that affect both the insect’s mass rearing, and the animals feed process. Therefore, in the current study the assessment of oxidative stress parameters of the black soldier fly (BSF) were investigated to quantify the deleterious effect of malathion-contaminated kitchen waste (1:1 vegetable: fruit waste) container on the insect. The different developmental stages of insect (adult and larva) were exposed to different concentrations (0, 0.005, 0.01, 0.015, and 0.02 mg/mL) of malathion. The results showed that the mean value of the reactive oxygen species (ROS), which included hydrogen peroxide (H2O2) and superoxide anion radicals (O2•-) concentrations were lower in larval stage than in adults, in all treated groups (0, 0.005, 0.01, 0.015, and 0.02 mg/mL malathion concentration). Also, the protein carbonyls amount and lipid peroxides levels were decreased in the 0.02 mg/mL Malathion compared to the control values. However, the cluster analysis revealed slight dissimilar patterns for control insects and the highest malathion concentration (0.02 mg/ml). These stage-related differences could occur from the different growth dynamic functions of larvae and adults. The larvae were distinguished by robust growth, and significant oxygen consumption. The results verified that oxidative stress parameters, especially protein carbonyls and α, α-diphenyl-β-picrylhydrazyl (DPPH) were promising, cheap, quick and cost-effective applications for determining the macromolecules damage, and antioxidant ability of H. illucens enclosed with malathion exposure. These findings described that malathion application induces macromolecules damage mediated through oxidative stress injury.

Non-enzymatic antioxidant response. The values of non-enzymatic antioxidant responses were represented in Fig. 3A and B. While DPPH recorded significantly higher value at 0.005 and 0.02 mg/ml malathion treated groups than the control group. In addition to the DPPH concentration of the control adult was significantly higher than all the treated adult groups (Fig. 3A). On the contrary, GSH concentration elevated significantly in all the treated larval groups, and in 0.01, and 0.02 mg/ml malathion adult groups compared to the control groups of H. illucens (Fig. 3B).

Relation and interaction assessment. Pearson′s correlation analysis between malathion concentration
and oxidative stress assays (ROS concentration, macromolecules damage, enzymatic and non-enzymatic anti- ) concentration (B). The data were expressed as mean ± SE. The concentrations were obtained from gut homogenates of 5th instar larvae and males of Hermetia illucens, which their food containers were exposure to different concentrations of malathion (0, 0.005, 0.01, 0.015, 0.02 mg/ ml). Mean values marked with the different lowercase letters were significantly different among malathion concentration assessment (ANOVA test Tukey's-b, Post Hoc test, p < 0.05). Table 1. The macromolecules oxidative damage in form of amount of protein carbonyls (OD/ mg protein), and concentration of lipid peroxides (mM cumene hydroperoxide/ mg protein). The protein carbonyls and lipid peroxides concentrations were obtained from gut homogenates of 5th instar larvae and males of Hermetia illucens, which their food containers were exposure to different concentration of malathion (0, 0.005, 0.01, 0.015, 0.02 mg/ml). The data were expressed as mean ± SE. Mean values marked with the different lowercase letters were significantly different among malathion concentration assessment (ANOVA test Tukey's-b, Post Hoc test, p < 0.05).  www.nature.com/scientificreports/ oxidant assays), revealed the most significant relationship in both larvae and adult stages ( Table 2). In larval stage, there was a correlation at p < 0.001 between malathion concentration and H 2 O 2 , PPO and GSH. Also, the strong correlation at p < 0.05 level was observed between malathion concentration (0-0.02 mg/ml) and SOD or CAT. Yet, there was no significant correlation between malathion concentration at adult stage, except for SOD at p < 0.001, and PPO, DPPH and GSH at p < 0.05 level (Table 2). However, the GEE interaction analysis between malathion concentration, developmental stage, interaction and intercept showed a significant influence between these factors in all oxidative stress parameters except in case of developmental stage effect on the levels of DPPH (Table 3). The Dendrogram of the cluster analysis, using Ward′s Method, revealed slightly dissimilar patterns for control insect groups and 0.02 mg/ml malathion concentration. The clustering oxidative stress assessment and antioxidant response of both larval and adult stages were shown in Fig. 4a-d. The level of oxidative stress assessment was highly similar in larval stage at 0, 0.005, and 0.01 mg/ml malathion concentration (Fig. 4a) however, the cluster of ROS and macromolecules damage occurred in adult stage at 0, 0.005, 0.01, and 0.015 mg/ml malathion  . Activity of non-enzymatic response in form of the concentration of anti-radical 2,2-diphenyl-1picrylhydrazyl (DPPH) (percentage of inhibition) (A), and antioxidant glutathione reduced (GSH) (µg/ mg protein) (B). The non-enzymatic antioxidants concentration was expressed, in a box-plot graph, as median, min., max., P25, and p75 values. The non-enzymatic antioxidant concentration obtained from gut homogenates of 5th instar larvae and males of Hermetia illucens, which their food containers were exposure different concentration of malathion (0, 0.005, 0.01, 0.015, 0.02 mg/ml). Mean values marked with the different lowercase letters were significantly different among malathion concentration assessment (ANOVA test Tukey's-b, Post Hoc test, p < 0.05). www.nature.com/scientificreports/ (Fig. 4b). The 0, 0.005, and 0.01 mg/ml malathion concentration created a separate cluster in antioxidant response system of larval H. illucens (Fig. 4c), though, in adult stage 0, 0.005, and 0.015 mg/ml malathion formed a separate cluster in antioxidant enzymatic and non-enzymatic response in gut homogenates of H. illucens (Fig. 4d). Environmental computing of the effect of different malathion concentration (0, 0.005, 0.01, 0.015, and 0.02 mg/ml) and developmental stage (5th larval instar and adult male) were shown in Fig. 5a-d. These effects were assessed in the form of principal component analysis (PCA). The PCA was done through variance covariance matrix analysis with two different components. Also, the eigen value tended to be dependent on 2 variables which were classified into first component and second component. The 1st component is different malathion concentration and the 2nd one is developmental stage. The normalization rotation method revealed that the O 2 •production rate, in groups treated with malathion concentration from 0 to 0.01 mg/ml, had a great ROS levels in larval and adult stages (Fig. 5a). Meanwhile, the macromolecules damage, in the form of protein carbonyls and lipid peroxide, was detected at concentration 0 or 0.02 mg/ ml malathion (Fig. 5b). The variance-covariance analysis showed a high variability of enzymatic antioxidant system along malathion concentration (Fig. 5c). However, the first component of PCA tended to be localized centric and reflected between non-enzymatic response and malathion concentration (Fig. 5d).

Discussion
Many studies focused on the role of H. illucens in organic waste recycling process, and the insect's valuable products, such as chitin, lipids, and proteins, which could be used on various industrial applications [25][26][27][28][29][30][31][32] . The modern scientific researches focused on using BSF larvae, that fed on organic waste, as animal feed. These wastes might contain different kinds and concentrations of pesticides which affect both the insect bioreactor and the feeding animals. The new stress problems such as the pesticides application or the mechanisms beyond the accumulation process were considered as one of the most interesting research points which focused on the phenomena of hormesis, adaptation, and mitigation 33 . Moreover, the lethargic effect of humans on the environmental components led to a dangerous and even lethal, backlash from ecosystem especially living organisms 34 . Several of the previous studies approved the assessment of oxidative stress in the biomonitoring of environmental pollution [35][36][37] . Exposure to the pesticides may raise the oxidative stress status directly by increasing the reactive oxygen species (ROS) over generation rate of the non-enzymatic and enzymatic antioxidants in the cells 36,38 . Maintaining the normal levels of oxidants in the cells is of utmost priority to avoid the negative actions of oxidative stress products, such as protein carbonyls and lipid peroxides levels on the living organisms' health 36,39 . Organophosphates could initiate ROS production and oxidation products in the cells 40,41 . malathion may indirectly increase the production of ROS inside the cells through disrupting the respiratory metabolism 42 , It also contains the P-S bond ("thion") that may convert to P-O bond ("oxon"), by the action of a microsomal system Table 2. Pearson′s correlation coefficient to detect the effect of malathion different concentration (0, 0.005, 0.01, 0.015, and 0.02 mg/ml) on reactive oxygen species concentration (ROS), (in form of hydrogen peroxide (H 2 O 2 ), superoxide anion radical (O 2 •-)), macromolecules damage (in form of protein carbonyls amount and lipid peroxides concentration), enzymatic antioxidant response (inform of superoxide dismutase (SOD), catalase (CAT), and polyphenol oxidase (PPO)), and non-enzymatic antioxidant response (inform of anti-radical 2,2-diphenyl-1-picrylhydrazyl (DPPH) and antioxidant glutathione reduced (GSH)) in the gut homogenates of 5th instar larvae and males of Hermetia illucens. * significant at p < 0.05; ** significant at p < 0.001. www.nature.com/scientificreports/ of enzymes named mixed-function oxidases (MFO), and cytochrome P450 (CYP450) 43 . The oxon compounds are highly toxic and can initiate oxidative damage to the living cells. malathion has a toxic effect on pupa, male and female of the peach fruit fly, Bactrocera zonata, with a higher resistance ratio in the field population than in laboratory insect population 44 . In the current study, a slight increase in the H 2 O 2 and O 2 •concentration was observed in the MHA than MHL by using all malathion concentration course (0-0.02 mg/ml) (Fig. 1). This explained the oxidants accumulation possibility 38 or the antioxidants levels' depletion in the adult stage than in the immature stage 36 . However, the H 2 O 2 concentration in 120th generation old of Spodoptera exigua didn't  www.nature.com/scientificreports/ change after the exposure to 44 μg/g of dry weight Cd 45 . The efficiency of the elimination process of oxidants and oxidative products may be impaired in the stressful conditions. This phenomenon occurred in this study where, the ROS, as O 2 •production rate, of the MHA increased than MHL (Fig. 1b). Also, H. illucens can normalize the concentration of H 2 O 2 and O 2 •especially in MHA. However, when the MHL was exposed to high concentration of malathion (0.02 mg/ml), the concentration of H 2 O 2 increased significantly than control values (Fig. 1a). Similarly, the toxicity of OP compounds induced some oxidative stresses, like elevation in the protein carbonyls amount or lipid peroxides level in some living organisms 46,47 , and caused physiological and pathological changes in tissues 48 . The LC 50 value of methidathion pesticide could affect the malondialdehyde level and antioxidant enzyme activities in the gut tissues of Lymantria dispar (Lepidoptera) larvae 49 . Similarly, our results recorded the highest concentration of protein carbonyls amount at 0.015 mg/ml malathion in the MHL, and the highest lipid peroxides concentration in MHL, and MHA at the same malathion concentration (Table 1). Yet, there was no significant difference between the highest concentration of malathion (0.02 mg/ml) and control (0 mg/ml) in both MHL and MHA (Table 1). This may be due to the action of enzymatic and non-enzymatic response 36 . The current study detected fluctuations in the concentration of protein carbonyls throughout the malathion concentration-course (Table 1), and this may reflect on the fluctuating homeostatic mechanism balance between protein degradation and production of protein carbonyls 36,50 . Lipid peroxidation can disrupt the membrane of the polyunsaturated phospholipids bilayer structure and function 51 . Also, products of lipid peroxidation are capable of disrupting conformations of many cellular proteins, including enzymes, by forming cross links with these proteins, inactivating their functions 52 . Lipid peroxidation is considered as a chain reaction; it produces lipid radical, lipid peroxyl radical, and then lipid hydroperoxide. This reaction can be stopped by termination www.nature.com/scientificreports/ reactions, such as the recombination of lipid peroxyl radicals and by a reaction with glutathione catalyzed by peroxidase 53 . Therefore, the pattern of fluctuation of lipid peroxides in H. illucens, formed post treatment with different concentration of Malathion, may be due to unbalanced levels of lipid peroxides production and their repairing mechanisms that may include antioxidant enzymes. Generally cited that, malathion is considered as neurotoxic component that inhibits the neuronal cholinesterase enzyme activity. Also, the peroxidative effects of OP were studied on the activities of antioxidant enzymes, and on lipid peroxidation in-vitro 54 , and in-vivo studies 55 . malathion treatment resulted in the elevation of lipid peroxidation concentration which was considered as an indicator of oxidative stress induction 56 . The results showed that the malathion applications led to elevation in the activities of the key antioxidant enzymes, SOD and CAT, over the constitutive levels except for different cases of malathion concentration ( Fig. 2a and b). The observed elevation seemed to occur in concomitance with the oxidative damages of the macromolecules; and may be in response to the formation of ROS as a consequence to the treated stressor 36 . SOD and CAT have a primary role in the oxidative stress defense through ROS elimination 53 . However, the significant depletion of PPO activity, which catalyzes the oxidation of phenolic compounds to quinones, occurred in both MHL and MHA treatments along concentration course of malathion (Fig. 2c). malathion treatment could acidify the medium and inhibit the PPO activity 57 . The DPPH results of MHL showed a significant increase in the 0.005 and 0.015 mg/ml malathion concentration treatment, compared to control values. Similarly, the malathion applications caused a serious risk to Saccharomyces cerevisiae (fungus). This in-vitro study showed that a flavonoid compound called naringin can inhibit some enzymes and can detoxify the DPPH radical 57 . Glutathione (GSH) acts as a redox factor to balance the redox state of the cell 58 . Reduced GSH is a chief cellular thiol element in the antioxidative system. www.nature.com/scientificreports/ Additionally, GSH and DPPH have a significant ROS scavenging role. The chemical stressors can increase the glutathione concentration in animals 59 . However, the one generation of Spodoptera exigua (Lepidoptera) which was exposed to Cd, didn't show elevation in the GSH concentration [60][61][62] . Our results showed that, the increase in GSH concentration was significant in both MHL and MHA, especially, at 0.015, 0.01 and 0.02 mg/ml malathion concentration, respectively (Fig. 3b). The interaction analysis, obtained from the computation of generalized estimating equation (GEE), revealed that the different concentration of malathion (0, 0.005, 0.01, 0.015, 0.02 mg/ml), the different developmental stage (MHL and MHA), and the interaction of these terms significantly influenced the physiological endpoints we measured (H 2 O 2 ,O 2 •-, protein carbonyls, lipid peroxides, SOD, CAT, PPO, DPPH, and GSH) (p value < 0.05), except for DPPH in the developmental stage effect (p value > 0.05) ( Table 3).
It has been concluded that the malathion exposure of insect food container can induce oxidative stress in the larval and adult male stages of H. illucens. The levels of ROS, macromolecules damage, enzymatic and nonenzymatic response in BSF to different malathion concentration may be used as a possible mechanism of malathion toxicity. The biochemical analysis of insects could be used as a novel strategy for assess the risk of pesticides accumulation on food container of recyclers of organic waste especially insects. Briefly, the tested hypothesis in this research has proved the ability of using oxidative stress parameters as bioindicator of malathion impact on the organic waste recycler, BSF. Meanwhile, malathion has uniform prooxidant properties in BSF, unlike the other phenolic compounds related to insecticides. The upcoming work will further investigate the fate of malathion in BSF, will answer a question about the role of the insect in reducing the toxicity and severity of malathion, and will shed more light on its role in bioremediation.

Materials and methods
Insects rearing and Malathion application. A colony of H. illucens was supplied from Al Qalyobia governorate and was reared under laboratory conditions for several generations, in the Department of Entomology, Faculty of Science, Cairo University. The experiments were made at summertime 2020, where the rearing conditions were (14:10 L:D; 34˚ ± 2; 60% RH) for adults and (0:24 L:D; 34˚ ± 2; 75% RH) for larvae. The insects were kept in mesh cages 30*30*40 cm 3 for adults (100 adults/ cage), and 20*20*10 cm 3 for larvae (200 larvae/ cage). Larvae were supplied daily with kitchen waste, 1:1 vegetable: fruit waste, (1000 larvae/ one kg kitchen waste) from household located at Giza Governorate, while adults were hydrated with water and sugar 24 .
The malathion application was performed by immersing the insect food container with different malathion concentrations (0, 0.005, 0.01, 0.015, or 0.02 mg/ml) for 24 h. Simultaneously, control insects were treated with immersing food container with distilled water. The levels of oxidative stress parameters of control insects were taken 100% levels. The range of low-level insecticide contamination of malathion were applied. Insects were divided into 2 groups: 5th larval instar, and male adult. The adult experiment specimens were taken into account the male's not females insects in order to avoid the compounding effects of ovarian development in the female insects. The sex of adult specimens was differentiating according to the insect morphological characters; where the males are characterized by a rounded genital apparatus. Each group was divided into 5 sub-groups of 250 individuals, which were exposed to malathion (0, 0.005, 0.01, 0.015, or 0.02 mg/ml) for 24 h post application. For each sub-group, 50 insects were dissected, after 24 h' malathion application, to isolate gut tissues. About 7.5 gm gut tissues, of each experimental sub-group, were homogenate in 7.5 ml ice-cold phosphate buffer (50 mM; pH 7.0 contained, 1 ml of 0.1% Triton X-100, 1 ml of 0.05 mM CaCl 2 ); and were centrifuged at 2000 × g for 10 min at 4 °C. The clear sample were stored at − 20 °C until use for further analysis. Each experiment was replicated three times.
The concentration of ROS. The concentration of H 2 O 2 was determined spectrophotometrically according to the method of Junglee et al. 63 . Briefly, using one step extraction-colorimetric procedure in which, homogenization step using PBS, pH = 7.0 mixed with 0.25 ml Trichloroacetic acid (TCA) (0.1% (w:v)), 0.5 ml KI (1 M), then the 1 ml samples were centrifuged at 12,000 × g for 15 min at 4 °C, and finally, the absorbance was measured at 240 nm. For the superoxide anion radical (O 2 •-) production rate of samples was determined using colorimetric analysis according to the method of Chen and Li 64 . The reaction mixture contains 0.25 mL epinephrine (1 mM), 0.25 mL NADPH (1 mM), 0.5 mL sodium phosphate buffer (PBS) (50 mM; pH 7.0), and 1 mL of the samples. The level of superoxide anion radical was determined by the rate of conversion of epinephrine to adrenochrome with 1 mM NADPH as substrate. The absorbance difference (A 485 -A 575 ) was recorded.
Oxidative damage assay. Protein carbonyls amount assay was performed according to procedure from Levine et al. 65 . After homogenization and centrifugation steps, a mixture of 800 µL sample, and 200 µl 2, 4-dinitrophenyl hydrazine (DNPH) (10 mM) was incubated for 30 min at room temperature, then precipitated with 1 ml TCA (1%). The pellet was washed four times with 1 ml absolute ethanol/ethyl acetate (1:1) mixture and dissolved in 1 mL of PBS (50 mM; pH 7.0), before being measured at 366 nm.
The lipid peroxides concentration was measured according to Hermes-Lima et al. 66 . After homogenization and centrifugation step, a mixture of 200 µl sample, 400 µl FeSO 4 (1 mM), 200 µl H 2 SO 4 (0.25 M) , and 200 µl xylenol orange (1 mM) were added, then absorbance measured at 580 nm. The mixture was incubated in the dark for 3 h at room temperature, and finally the absorbance re-measured at 580 nm after adding of 10 µl cumene hydroperoxides (0.05 mM) (as an internal standard). The change in absorbance due to addition of internal standard was calculated.
Antioxidant enzymatic response. SOD activity was measured based on the procedure described by Misra  www.nature.com/scientificreports/ 10.0), 35 µl of EDTA (10 mM), 87 µl of the sample and 0.5 ml of freshly prepared epinephrine (15 mM). The absorbance was measured at 480 nm. The activity of CAT was assessed in compliance with the method of Aebi 68 . The reaction mixture contained 0.9 ml of a potassium phosphate buffer (50 mM, pH 7.0), 60 µl of the sample and 40 µl of freshly prepared H 2 O 2 (10 mM). The change in absorbance was measured at 240 nm over a period of 0.5 min. The method of Kumar and Khan 69 was used to assess the PPO activity in a reaction mixture containing 0.9 ml of a potassium phosphate buffer (50 mM, pH 7.0), 0.25 ml of 0.1 M catechol and 0.25 mL sample. The reaction formed purpurogallin which was measured at 495 nm.
Antioxidant non-enzymatic response. DPPH antioxidant activity was determined according to Blois 70 , by adding 0.5 ml DPPH (o.5 M) to 0.5 ml sample and incubated for 20 min before measuring absorbance at 525 nm. DPPH assay was based on the scavenging capability measurement. The nitrogen atom contains an odd electron which is reduced by delivering a hydrogen atom from antioxidants to hydrazine. The procedure of Allen et al. 71 was adapted for determining GSH concentration. Briefly, the reaction mixture, containing 150 μl sample, 800 μl PBS (50 mM; pH 8.0), and 50 μl 5, 5´-Diothio bis-2-nitrobenzoic acid (2 mM), then incubated at 25 °C for 20 min. The absorbance of the reaction mixture was 412 nm. The GSH content was determined from a GSH standard curve. The total protein concentration of samples was determined spectrophotometrically according to the method of Bradford 72 . Briefly, 0.9 mL of the Coomassie brilliant blue (0.5 mM) were mixed with 0.1 mL sample and incubated at room temperature for 2 min. The OD of the protein sample was measured at 595 nm.
Statistical analysis, relation, and interaction assessment. Statistical analysis was performed using IBM SPSS Statistics for Windows (Version 17.0. Armonk, NY: IBM Corp.). A parametric test was carried out using ANOVA test Tukey's-b, Post Hoc test for assessment the malathion concentration effect and T-test, for insect developmental stage. Correlations between malathion concentration and the experimental assays of oxidative stress parameters, including, ROS, macromolecules damage, enzymatic antioxidant response and nonenzymatic antioxidant response, were performed based on Pearson's regression analysis using linear regression models. Hierarchical Cluster Analysis (HACA) based on agglomerative statistics using Ward′s Method was calculated for oxidative stress parameters. The goal of HACA is to find possible clusters or groups among the observational units, based on level of similarities and differences 12 . Generalized Estimating Equation (GEE) was used to examine the effect of malathion concentration, insect developmental stage, combined effect of concentration and developmental stage, and finally intercept on the parameters of oxidative stress. The principal component analysis (PCA) was performed the possible assessment of different malathion concentration and developmental stage on the oxidative stress parameters of BSF.
Ethical approval and consent to participate. This article does not contain any studies with human participants or animals that require ethical approval.